This invention is in the field of molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding scorpion toxins that are potassium channel modifiers.
Scorpion venoms have been recognized as a source of peptidyl inhibitors of various types of potassium ion (K) channels. Some of these peptides have been purified to homogeneity and their properties characterized. The most extensively studied of these toxins is charybdotoxin (ChTX). ChTX is a thirty-seven amino acid peptide isolated from venom of the old world scorpion Leiurus quinquetriatus var. hebraeus. Originally described as an inhibitor of the high-conductance, Ca+2-activated K (Maxi-K) channel present in muscle and neuro-endocrine cells, ChTX was later found to also inhibit a number of different medium- and small-conductance Ca+2-activated K-channels, as well as a voltage-dependent K-channel (K(v) 1.3). In each case, channel inhibition occurs with similar potency, in the low nanomolar range. A related toxin, iberiotoxin (IbTX), shares 68% sequence homology with ChTX and selectively blocks the Maxi-K channel. Other peptidyl inhibitors, such as limbatustoxin (LbTX) and kaliotoxin (KTX), have also been shown to possess greater selectivity for the Maxi-K channel. Other peptidyl toxins homologous to ChTX have been identified (e.g., noxiustoxin).
Potassium channels modulate a number of cellular events such as muscle contraction, neuro-endocrine secretion, frequency and duration of action potentials, electrolyte homeostasis, and resting membrane potential. These channels comprise a family of proteins that have been classified according to their biophysical and pharmacological characteristics. Inhibition of K-channels, in their role as modulators of the plasma membrane potential in human T-lymphocytes, has been postulated to play a role in eliciting immunosuppressive responses. In regulating membrane potential, K-channels play a role in the regulation of intracellular Ca+2 homeostasis, which has been found to be important in T-cell activation.
Potassium channel modifiers are small polypeptides (31 to 37 amino acids) which form compact structures kept rigid by three disulfide bridges. Use of synthetic analogs with point mutations has determined that single amino acids residues are important for receptor binding and for biological activity of K-channel toxins (Sabatier et al. (1994) Int. J. Peptide Protein Res. 43:486-495). Moreover, a drug with high affinity for the receptor could be expected to produce irreversible blockade of synaptic transmission. When labeled with a tracer molecule, such a drug would provide a reliable way of tagging receptors to permit measurement of their number and distribution within cells and tissues. These features would have very valuable consequences for research on excitatory amino acid neurotransmission and for the development of therapeutic agents to treat central nervous system dysfunction in humans and animals. Methods for treating heart and neurological diseases by applying toxins derived from spiders have been described (U.S. Pat. No. 4,925,664).
Arthropod animals, including insects, and certain parasitic worms, use excitatory amino acids as a major chemical neurotransmitter at their neuromuscular junction and in their central nervous system. Because of the damage done by insect pests and the prevalence of parasitic worm infections in animals and humans in many countries, there is a constant need for potent and specific new pesticides and anthelmintic drugs that are non-toxic to humans, pets, and farm animals.
Many arthropods produce a mixture of insecticidal proteins referred to as venom. These toxic substances are synthesized in specialized glandular tissues, which, when directed by a stinging or piercing apparatus, are capable of paralyzing the arthropod""s prey. Small, slow moving or stationary arthropods have adapted a strategy to instantaneously paralyze their prey by utilizing neurotoxic components of the venom at very low concentrations. These components, or neurotoxins, interfere with the function of insect nervous tissues through efficient competition for certain receptor sites. Many of these neurotoxins are polypeptides. These have been divided into different classes based on their host specificity and mode of action (Zlotkin (1991) Phytoparasitica 19:177-182). For example, neurotoxic peptides isolated from numerous species of scorpions have been divided into classes that affect arthropods and classes that affect mammals.
Due to a combination of problems associated with some synthetic insecticides, including toxicity, environmental hazards, and loss of efficacy due to resistance, there exists a continuing need for the development of novel means of invertebrate control, including the development of genetically engineered recombinant baculoviruses which express protein toxins capable of incapacitating the host more rapidly than the baculovirus infection per se.
Many different toxins have been isolated from scorpions. Cobatoxin 1 and 2 are potassium channel blocking toxins isolated from scorpions and which have 32 amino acids and contain 3 disulfide bridges (Selisko et al. (1998) Eur. J. Biochem. 254:468-479). Isolated from scorpion venom, the kaliotoxin 2 precursor contains a 22 amino acid signal sequence and a 37 amino acid mature peptide which specifically binds to receptor sites in rat synaptosomes (Laraba-Djebari et al. (1994) J. Biol. Chem. 269:32835-32843). Leiuropeptides I, II and III are peptides with cysteine pattern analogous to that of short-chain scorpion toxins. Leiuropeptide I acts on potassium channels, has 31 amino acids and a positively charged region that binds to receptors (Buisine E. et al. (1997) J. Pept. Res. 49:545-555). Leiurotoxin I is a 31 amino acid peptide with three disulfide bridges holding the amino-terminal alpha structure on the side of the carboxy-terminal two beta barrels (Martins et al. (1990) FEBS Lett. 260:249-253). The 35 amino acid neurotoxin P2 from the Androctonus mauretanicus scorpion is a structural homologue of the so called Buthus epeus insect toxins (Rosso and Rochat (1985) Toxicon 23:113-125). Having 36 residues and some overall homology to charybdotoxin and noxiustoxin, toxin 15-1 blocks calcium-activated potassium currents in muscle fibers (Marshall (1994) Toxicon 32:1433-1443).
Scorpion venoms have been identified as possible sources of compounds providing insecticidal properties. Two insect-selective toxins isolated from the venom of the scorpion Leiurus quinquestriatus and affecting sodium conductance have been reported previously (Zlotkin et al. (1985) Arch. Biochem. Biophys. 240:877-87). One toxin, AaIT, induced fast excitatory contractive paralysis of fly larvae and the other, LqhIT2, induced slow depressant flaccid paralysis suggesting that these two toxins have different chemical and pharmacological properties (Zlotkin et al. (1971) Biochimie (Paris), 53:1073-1078). Thus, other toxins derived from scorpion venom will also have different chemical and pharmacological properties.
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence of at least 81 nucleotides selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19; (b) a second nucleotide sequence encoding a polypeptide of at least 27 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; or (c) a third nucleotide sequence comprising the complement of the first or second nucleotide sequences.
In a second embodiment, this invention relates to an isolated polynucleotide encoding a mature K-channel agonist.
In a third embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least one of 30 (preferably at least one of 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and the complement of such nucleotide sequences.
In a fourth embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.
In a fifth embodiment, the present invention concerns a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, mammalian cell or an insect cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
In a sixth embodiment, the invention also relates to a process for producing a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting a compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
In a seventh embodiment, the invention concerns a K-channel agonist polypeptide of at least 27 amino acids comprising at least 80% identity based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.
In an eighth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a scorpion K-channel agonist polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 30 (preferably at least 40, most preferably at least one of 60) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a scorpion K-channel agonist amino acid sequence.
In a tenth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a scorpion K-channel agonist polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing a cDNA or genomic fragment that comprises the isolated DNA clone.
In an eleventh embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide or an isolated polypeptide of the present invention.
In a twelfth embodiment, this invention concerns a method for expressing a gene encoding a scorpion K-channel agonist in the genome of a recombinant baculovirus in insect cell culture or in viable insects wherein said insect cells or insects have been genetically engineered to express a potassium channel blocking toxin 15-1, a Bmtx 1, a neurotoxin P2, a leiurotoxin I, a leiuropeptide I, a leiuropeptide III, a kaliotoxin 2 precursor or a cobatoxin 1. The recombinant baculovirus expression vector comprising a DNA sequence encoding a polypeptide of at least 27 amino acids comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.